1. Field of the Invention
This invention relates to a 3.alpha.-hydroxysteroid oxidase and also to a quantitative analysis of a 3.alpha.-hydroxysteroid making use of the 3a-hydroxysteroid oxidase.
2. Description of the Prior Art
Among 3.alpha.-hydroxysteroids in an organism, there are steroid hormones such as androsterone, besides bile acid and the like. Of these, bile acid has particularly important significance for clinical diagnoses. Bile acid is composed principally of glucocholic acid, taurocholic acid, glucochenodeoxycholic acid, taurochenodeoxycholic acid, glucodeoxycholic acid, taurodeoxycholic acid and the like. After having been synthesized from cholesterol in a liver, it is circulated through an extremely closed cycle called "enterohepatic circulation system". Bile acid is therefore contained only in a very trace amount in peripheral blood of healthy people. This cycle is however subjected to rhexis by a liver or biliary tract disease, resulting in an increased blood level of bile acid. Making use of such an increase in the blood level of bile acid as an index, it is therefore possible to diagnose such a liver or biliary tract disease and at the same time, to determine its graveness to a certain extent. For these reasons, it has become important to quantitatively analyze bile acid, which is one of 3.alpha.-hydroxysteroids, in an organism, especially, serum for the diagnosis of liver and/or biliary tract diseases.
As quantitative analyses of 3.alpha.-hydroxysteroids known to date, there are chromatography, enzyme assay, immunoassay, etc. In the field of routine clinical tests, enzyme assay is primarily used owing to its simplicity. Namely, 3.alpha.-hydroxysteroid dehydrogenase (hereinafter abbreviated as "3.alpha.-HSD") is caused to act on bile acid (a 3.alpha.-hydroxysteroid) in the presence of nicotinamide adenine dinucleotide (hereinafter abbreviated as "NAD"), thereby converting AND to reduced nicotinamide adenine dinucleotide (hereinafter abbreviated as "NADH"). Thereafter, its quantitative analysis is carried out by any one of the following methods:
(1) The fluorescence of the resultant NADH is measured.
(2) The resultant NADH is converted back to AND under the action of diaphorase and at the same time, coexisting resazurin is converted to resorufine. The fluorescence of the resorufine is then measured
(3) The resultant NADH is converted back to AND under the action of diaphorase and at the same time, coexisting nitroblue tetrazolium is converted to diformazan. The fluorescence of the diformazan is then subjected to colorimetry.
The above methods (1) and (2) are however fluorometric methods, and their procedures are complex and moreover they require expensive equipment. Under the circumstances, they are seldom relied upon in routine tests. On the other hand, the method (3) is poor in sensitivity. Moreover, it requires each sample in a large volume since the blood level of bile acid is extremely low. It is also prone to interference by other components in the sample. The method (3) is not therefore fully satisfactory. Further, the resulting diformazan is a substance having low water solubility and is hence accompanied by drawbacks such that it is adsorbed and is caused to precipitate on instruments employed upon measurement, e.g., cells.
It has therefore been desired to develop an advantageous method for the measurement of a 3.alpha.-hydroxysteroid, which is free of drawbacks such as those described above and uses an enzyme.